We have discovered a novel proline-driven conformational switch in the immunological protein kinase Interleukin-2 tyrosine kinase (Itk). Proline cis/trans isomerization within the Itk Src homology 2 (SH2) domain mediates conformer-specific recognition of Itk signaling partners and an important regulatory interaction with the ubiquitous peptidyl-prolyl isomerase cyclophilin A (CypA). This proposal is focused on clearly defining the role of proline isomerization in the Itk regulatory apparatus. Our extensive experience in working with Itk and CypA both at the structural and functional level places us in an ideal position to dissect the Itk regulatory mechanism. Proline isomerization is an intrinsic conformational exchange event that is most easily studied by NMR spectroscopy. Thus, our approach is anchored in structural studies that provide the indispensable atomic-level information required to fully examine the functions of these intracellular signaling proteins in T cells. Our structural insights have already been tested in mouse model systems providing further evidence that Itk regulation is linked to a single proline residue that is the target of CypA. Moreover, the proline isomerization event within Itk may represent another form of posttranslational 'modification' akin to protein phosphorylation. Like phosphorylated amino acid side chains, critical proline residues could control ligand binding and serve as recognition sites for regulatory partners such as the peptidyl-prolyl isomerases. Our detailed analysis of Itk and CypA will lay the groundwork for identification of other proline switches within the proteins that control immune cell signaling. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]